Confocal microscopy

ABSTRACT

An improved confocal microscope system is provided which images sections of tissue utilizing heterodyne detection. The system has a synthesized light source for producing a single beam of light of multiple, different wavelengths using multiple laser sources. The beam from the synthesized light source is split into an imaging beam and a reference beam. The phase of the reference beam is then modulated, while confocal optics scan and focus the imaging beam below the surface of the tissue and collect from the tissue returned light of the imaging beam. The returned light of the imaging beam and the modulated reference beam are combined into a return beam, such that they spatially overlap and interact to produce heterodyne components. The return beam is detected by a photodetector which converts the amplitude of the return beam into electrical signals in accordance with the heterodyne components. The signals are demodulated and processed to produce an image of the tissue section on a display. The system enables the numerical aperture of the confocal optics to be reduced without degrading the performance of the system.

DESCRIPTION

1. Field of the Invention

The present invention relates to confocal microscopy for examination ofobjects, such as biological tissue, and particularly to a confocalmicroscope system for scanning below the surface of tissue, whichutilizes heterodyne detection to produce confocal images of tissuesections. This invention is especially suitable for providing aninstrument for dermal or surgical pathology applications.

2. Background of the Invention

Confocal microscopy involves scanning a tissue to produce microscopicimages of a slice or section of tissue. Such microscopic imaged sectionsmay be made in-vivo and can image at cellular resolutions. Examples ofconfocal scanning microscopes are found in Milind Rajadhyaksha et al.,"In vivo Confocal Scanning Laser Microscopy of Human Skin: Melaninprovides strong contrast," The Journal of Investigative Dermatology,Volume 104, No. 6, June 1995, pages 1-7, and more recently, in MilindRajadhyaksha et al., "Confocal laser microscope images tissue in vivo,"Laser Focus World, February 1997, pages 119-127. These systems haveconfocal optics which direct light to tissue and image the returnedreflected light. Such confocal microscope systems can focus and resolvea narrow width of tissue as an imaged section, such that tissuestructures can be viewed at particular depths within the tissue, therebyavoiding evasive biopsy procedures for pathological examination of thetissue, or allow pathological examination of unprepared excised tissue.

Two parameters which effect the performance of confocal microscopesystems in imaging tissue sections are the numerical aperture (NA) ofthe optics and the wavelength of the beam scanned through the tissue.The axial resolution, i.e., the thickness of the imaged section, andlateral resolution of confocal microscope systems are directlyproportional to the wavelength of the light source and inverselyproportional to NA² (axial) and NA (lateral). In other words, the higherthe NA, the thinner the imaged section, while the lower the NA, thethicker the imaged section. Both the axial resolution and the lateralresolution are optimized in a confocal microscope system suitable forpathological examination to the dimensions of the tissue structures,such as cells, which are of interest. As discussed in the MilindRajadhyaksha et al. article appearing in Laser Focus World, February1997, the use of a near-infrared light source between about 700 nm and1200 nm and optics with a NA of about 0.7-0.9 have provided optimalresults for imaging tissue sections with sufficient discrimination ofcellular level structures. One problem with using optics providing NAvalues about this range is that they are large and expensive,particularly for the objective lens which focuses light into andcollects light from the tissue, and are very sensitive to aberrations,such as introduced by the object being imaged. Accordingly, it isdesirable to provide imaging of tissue sections in a confocal microscopeusing lower cost and smaller optics having a NA below 0.7 withoutsacrificing imaging performance, in particular depth discrimination andscattered light rejection.

Accordingly, it is a feature of the present invention to improveconfocal microscopy by combining the depth response of confocal imagingwith the coherence function of heterodyne detection using a synthesizedbeam of multiple wavelengths of light, such that lower NA confocaloptics and inexpensive laser diode sources may be used. Heterodynedetection has been proposed for imaging in U.S. Pat. No. 5,459,570,which describes an apparatus using an optical coherence domainreflectometer for providing images of a tissue sample to perform opticalmeasurements. However, this apparatus is limited in depth resolution anddoes not utilize confocal optics for microscopic imaging. Other opticalsystems have used multiple wavelengths of light, but are limited togenerating interference patterns for visualizing fringes characterizingthe surface of objects, such as shown in U.S. Pat. No. 5,452,088, whichdescribes a multi-mode laser apparatus for eliminating backgroundinterference, and U.S. Pat. No. 4,632,554, which describes a multiplefrequency laser interference microscope for viewing refractive indexvariations. Such interferometric-based optical systems have no confocaloptics or heterodyne detection, nor do they provide imaging within atissue sample. A confocal microscope using multiple wavelengths of lighthas been proposed in U.S. Pat. No. 4,965,441, but this microscope islimited to focusing at different altitudes for surface examination of anobject and does not have heterodyne detection.

SUMMARY OF THE INVENTION

It is the principal object of the present invention to provide animproved confocal microscopy method and confocal microscope system forimaging sections of tissue using heterodyne detection.

It is another object of the present invention to provide an improvedconfocal microscope system for imaging tissue using a synthesized lightsource to produce a beam having different wavelengths, in which thesynthesized light source combines beams from multiple light sourcesproducing light at each of the different wavelengths.

It is another object of the present invention to provide an improvedconfocal microscope system for imaging which can use low NA confocaloptics, such as below 0.7, while achieving imaging performance in termsof axial resolution equivalent to prior art confocal microscope systemsusing higher NA confocal optics, such as between 0.7 and 0.9.

Briefly described, the system embodying the present invention includes asynthesized light source for producing a single beam of light ofmultiple, different wavelengths from multiple laser sources, and a firstbeam splitter for separating the single beam into an imaging beam and areference beam. The phase of the reference beam is modulated by anoptical modulator, while confocal optics scan and focus the imaging beambelow the surface of the tissue and collect returned light of theimaging beam from the tissue. A second beam splitter is provided forinteracting the returned light of the imaging beam with the modulatedreference beam to provide a combined return beam having heterodynecomponents. The heterodyne components in the return beam represent thespatial overlapping of the imaging and reference beams over thebandwidth of the different wavelengths produced by the synthesized lightsource. The return beam is received by a photodetector which convertsthe amplitude of the light of the return beam into electrical signals inaccordance with such heterodyne components representative of the tissuesection. The electrical signals are then processed by a controller, suchas a computer, to produce an image of the tissue section on a displaycoupled to the controller.

To promote the interaction of the imaging and reference beams in thereturn beam, the path lengths of the imaging and reference beams arematched such that the difference between their path lengths areapproximately equal to integer multiples of the separation of the peaksin the coherence function produced by the synthesized light source.

The performance of the system, in terms of the axial resolution of theimaged tissue section, depends on the numerical aperture (NA) of theconfocal optics and the multiple, different wavelengths of the beamproduced by the synthesized light source, such that lower NA optics canbe used to provide an axial resolution previously afforded by confocalmicroscope systems using higher NA optics between 0.7 and 0.9.

The system improves confocal microscopy by combining the axialresolution of confocal detection and the axial ranging of heterodynedetection of light with a coherence function, that is preferablyperiodic, to provide an axial (depth) resolution that is an improvementover that provided by confocal or heterodyne detection alone. It isbelieved that the heterodyne components are produced by overlapping oneof the peaks of the coherence function with the broader depth responseof the confocal optics, while all the other peaks do not contribute tothe image of the tissue due to suppression by the confocal depthresponse. The signal is spatially limited to a region of said tissue inthe focal plane along which the confocal optics scan and focus theimaging beam in the tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing objects, features and advantages of the invention willbecome more apparent from a reading of the following description inconnection with the accompanying drawings in which:

FIG. 1 is a block diagram of the system in accordance with the presentinvention;

FIG. 2 is a block diagram of the synthesized light source of FIG. 1; and

FIG. 3 is a graph showing an example of the axial resolution of thesystem of FIG. 1 in terms of depth response.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, a confocal microscope system 10 is shown forproducing images of sections of an object 12, such as a tissue sample orin-vivo tissue of patient, below the surface of the object. System 10includes a synthesized light source 14 providing a single beam 15 havingseveral, different wavelengths of light. Synthesized light source 14includes a number (N) of multiple light sources each providing lightbeams at a different wavelength, which are combined into a single beam15. Beam 15 thus represents light that has a coherence function withnarrow peaks depending on the wavelengths (or frequencies) of each ofthe multiple light sources of synthesized light source 14. Thewavelengths of the light sources of the synthesized light source areselected to be transparent to object 12 to a particular depth from theobject's surface. For tissue, such transparency occurs in the infraredspectrum of light.

An example of synthesized light source 14 with four light sources (N=4)is shown in FIG. 2 in which the light sources are represented by lasermodules 16. The four laser modules 16 each provide a beam at a differentwavelength (or band) λ₁, λ₂, λ₃, and λ₄, respectively. Preferably, thewavelengths satisfy the equation λ₄ <λ₃ <λ₂ <λ₁ and the wavelengths areequally separate from each other, such as at 80 nm intervals in therange of 700 nm to 1200 nm. Each beam is successively combined bydichroic beam splitters 18 to provide a single beam 15 of discretewavelengths (or bands) λ₁, λ₂, λ₃, and λ₄. Specifically, the beam (λ₁)from laser modules 16a and the beam (λ₂) from laser module 16b arecombined by beam splitter 18a. The beam (λ₁, λ₂) from beam splitter 18ais then combined by a beam splitter 18b with the beam (λ₃) from lasermodule 16c. The beam (λ₁, λ₂, λ₃) from beam splitter 18b is nextcombined with the beam (λ₄) from laser module 16d to provide beam 15(λ₁, λ₂, λ₃, λ₄). Any number of beams may be combined in this manner.Each beam splitter 18 maximally transmits each wavelength, for example,beam splitter 18c may have transmission greater than 95% for eachwavelength band λ₁, λ₂, λ₃. Similarly, each beam splitter reflectsmaximally the wavelength to be combined. For example, beam splitter 18creflects λ₄ with reflectivity greater than 95%. The laser modules 16 mayuse diode lasers to produce a beam of high brightness which are eitherfixed, or tunable, to a desired wavelength, and have sufficiently largecoherence lengths, such as greater than 1 mm, and may be for example thefollowing commercially available laser diodes Toshiba TOLD9215, SanyoSDL3034, Sanyo SDL4032, and EG&G C86136, C86125E.

Returning to FIG. 1, beam 15 from synthesized light source 14 is splitby a beam splitter 20 into an imaging beam 22, which travels along animage arm 23 of system 10, and a reference beam 24, which travels alonga reference arm 25 of the system. Beam splitter 20 may be anon-polarizing beam splitter having similar optical transmissionproperties for each of the different wavelengths of synthesized lightsource 14, so that the merging of each wavelength is combined additivelyand linearly. For example, if synthesized light source 14 has two lightsources, beam splitter 20 would be approximately 50/50 (equallytransmissive) for the wavelength band of each source.

The imaging beam 22, passing through a beam splitter 32, is incident ona scanning device 26 for deflecting the beam 22 into a scanning beam 28that is focused by an objective lens 30 into object 12. The multiplewavelength imaging beam is thus scanned and focused to a polychromaticspot at a depth into the object (i.e., below the surface of the object).The scanning device 26 may be a typical scanning mechanism fortwo-dimensional imaging, such as a rotating polygon mirror for scanningin a first direction, and a galvomirror which deflects the beam in asecond direction. Other scanning mechanisms may also be used, such asone or two positionable galvomirrors. A relay mirror 29 may be providedto deflect the scanning beam to objective lens 30.

The imaging beam is returned from the surface or internal section of theobject 12 to be imaged. Objective lens 30 collects the reflected lightof the imaging beam 22 from the object 12, and such collected light isdeflected via relay mirror 29 and scanning device 26 to beam splitter32.

The reference beam 24 is transmitted, via relay mirror 27, to a phasemodulator 34, such as an acousto-optic modulator, which is operated by asignal from a CW oscillator 35, to modulate beam 24, and hence eachwavelength of the beam, by a certain frequency produced by oscillator35, such as 100 MHz. The frequency of the phase modulation does not varyduring scanning by scanning device 26, and should be greater than thescan frequency, i.e., the velocity of the scanning beam at the objectdivided by the lateral resolution. Alternatively, the acousto-opticmodulator may be replaced by a piezo-actuated moving mirror whichproduces a frequency (Doppler) shift in the reference beam, or by anelectro-optical modulator. The modulated reference beam 24a isdeflected, via relay mirror 31, to a beam splitter 32. At beam splitter32, the modulated reference beam 24a and the imaging beam lightreturning from the object 12 are combined into a return beam 36, so thatthe modulated reference beam and the imaging beam returned lightspatially overlap and interact to produce heterodyne components in thereturn beam 36. Beam splitter 32 may be a non-polarizing beam splitterhaving similar optical transmission properties for the differentwavelength bands of light from synthesized light source 14.

The return beam 36 is incident on a photodetector 38. Photodetector 38may consist of a single photo-diode detector 37a and a confocal aperture37b, such as a pinhole, in the path of return beam 36. Aperture 37breduces the amount of stray light that falls on detector 37a. It isbelieved that spatial limiting (filtering) detection to a particularregion in the focal plane of the scanned imaging beam may be provided bythe use of heterodyne detection in which the reference beam acts as anaperture for the returned light of the imaging beam from the object.Alternatively, or in combination, aperture 37b provides for such spatialfiltering. Photodetector 38 may also be accomplished by a lens (notshown) which has a focal power which overfills photo-diode 37a, insteadof confocal aperture 37b. The synthesized light source 14, beamsplitters 20 and 32, scanning device 26, objective lens 30, andphotodetector 38, represent the confocal optics of system 10, and may besimilar to those used by a typical confocal imaging system, except thata synthesized light source is used rather than conventionalmonochromatic light source illumination.

Photodetector 37a of the photodetector assembly 38 produces anelectrical signal 39 in response to the amplitude of the light incidentupon the photodetector in the range of the different wavelengths of thesynthesized light source. Signal 39 is inputted to a demodulator 40,which operates at the same modulating frequency as phase modulator 34 todemodulate the signal, such that the electrical output signal 42 fromdemodulator 40 is directly proportional to the amplitude of themodulation in signal 39. Demodulator 40 may operate via an input signalfrom oscillator 35, if needed, however the demodulator is not sensitiveto the phase of signal 39. For example, demodulator 40 may operate bytypical amplitude demodulation (similar to demodulation of an AM radio),or demodulation as described in U.S. Pat. No. 5,459,570.

A controller 44 is provided in system 10 which receives the outputsignal 42 from the demodulator and generates an image on a display (orCRT) 46 responsive to signal 42 as successive frames in real-time, inaccordance with the scanning pattern of scanning device 26. For example,controller 44 may sample signal 42 to acquire data represent successiveraster lines of an image correlated to the scanning mechanism as itscans the imaging beam successively across the object. Controller 44 mayalso enable and disable the operation of system 10 by controlling thelight source 14, scanning device 26, phase modulator 34, oscillator 35,and demodulator 40, via control lines not shown. Optionally, theposition of the scanning device 26 during the scan may be monitored orcontrolled by the controller 44. The controller may be a computer, suchas a PC, which uses typical display driving software for producingimages on display 46 coupled to the computer.

In system 10, the imaging beam 22 and reference beam 24 desirably areboth beams of collimated light. This may be provided by assuring thatbeam 15 from synthesized laser source 14 is collimated by the opticsahead of the beam splitter 20 before being split into the imaging andreference beams. If needed, a collimation telescope or lens may be usedwith the beam from each of the light sources of the synthesized lightsource 14 to achieve such collimation of beam 15. By using collimatedlight in system 10, the imaging and reference beam waves will have thesame phase curvature at photodetector 37a, such that they spatiallyoverlap and interact (by interference) with each other in the returnbeam 36 at the photodetector 37a to produce heterodyne components.

The imaging beam travels along the image arm path which represents thepath of the imaging beam 22 from beam splitter 20 to object 12 and thereturned light from the object to beam splitter 32, while the referencebeam travels along the reference arm path which represents the path frombeam splitter 20 through modulator 34 to beam splitter 32. The lengthsof the reference arm path and the image arm path are matched in system10 so that the imaging beam and reference beam interact to produceheterodyne components. Matching of the reference arm and image arm pathsoccurs when the difference in length of the two paths are approximatelyequal to integer multiples of the separation of the peaks of thecoherence function of the synthesized light source 15. This separationis dependent on each of the different wavelengths of light produced bythe synthesized light source. In the case of two light sources withinsynthesized light source 14, correction of any mismatch between thereference and image arm paths lengths may be achieved by turning off onelaser source first (which gives the confocal response) and then, withboth laser sources on, adjusting the reference arm length using theconfocal image on display 46 as a template. With more that twowavelengths the above procedure can be repeated for each wavelengthused.

The performance of system 10 in terms of axial resolution of the imagedtissue section depends on the number of light sources in the synthesizedlight source 14, the wavelengths of such light sources, and the NA ofthe confocal optics of the system. Table I below shows an example of theperformance of system 10 using the combination of any two (N=2) of fivedifferent light sources at wavelengths 670 nm, 780 nm, 830 nm, 905 nm,and 1050 nm, respectively. For each pair of wavelengths in a column androw of Table I, the optimum (or actual) NA of the confocal optics foruse with such wavelengths is first indicated, and then the benefit inaxial resolution provided is shown by the arrow to the NA which would berequired of the confocal optics to obtain such axial resolution at thelower of the two wavelengths.

                  TABLE I                                                         ______________________________________                                         (nm) 670    780       830     905     1050                                   ______________________________________                                        670   x      0.53 → 0.69                                                                      0.62 → 0.79                                                                    0.71 → 0.87                                                                    0.83 → 0.95                       780  x 0.35 → 0.48 0.52 → 0.69 0.71 → 0.87                                                     830   x 0.40 → 0.55 0.64                                              → 0.81                            905    x 0.52 → 0.69                                                   1050      x                                                                 ______________________________________                                    

For example, using two light sources in synthesized light source 14which operate at wavelengths 670 nm and 830 nm, respectively, andconfocal optics having an NA of 0.62, the axial resolution provided bythe system is the same as if such confocal optics had an NA of 0.7 g andthe synthesized light source where substituted for a single light sourceat 670 nm. Optimal results in Table I, where the actual NA is the lowestand provides performance near the NA range of 0.7 to 0.9, occurs atcombinations of wavelengths 670 nm and 780 nm, 780 nm and 905 nm, and905 nm and 1050 nm. Accordingly, lower NA confocal optics can be used toproduce the same axial resolution afforded by higher NA confocal opticsin prior art single light source confocal microscope systems.Furthermore, if all five light sources of Table I were used in system10, the optimum NA of the confocal optics is 0.47 and the axialresolution provided by the depth response is equivalent using confocaloptics with an NA of 0.8 with only a single light source at 670 nm.

Preferably, only two laser sources are used to reduce the complexity ofthe system, however, more than two laser sources may be provided suchthat combinations of all or some of their wavelengths may be used toprovide the desired response of system 10. Thus, the illuminationprovided by the synthesized light source provides freedom in the choiceof the bandwidth over the different wavelengths of such illumination.

In imaging tissue sections by system 10, longitudinal chromaticaberrations may be corrected, if needed, by individually adjusting theposition of the focus for each wavelength produced by the synthesizedlight source. Spherical aberrations in the system, introduced primarilyby the sample, may be reduced by the use of index matching materialbetween the objective lens and the object, or by the use of an indexmatched immersion objective lens. The use of low NA confocal optics mayfurther reduce spherical aberration. Objective lens 30 may be either adry or immersion objective lens 30, although penetration depth of thescanning beam may be improved by the use of an immersion objective lens.

The following theoretical explanation is given in order to demonstratethe improvement obtained by means of heterodyne detection in accordancewith the invention. The presentation of the explanation does not implylimitation of the invention to any theory of operation. The explanationuses the following terms, equations and parameters presented below.

N is the number of light sources in the synthesized light source inwhich λ_(N) is the wavelength of the Nth source. For example, λ₁ is thewavelength of the first source, and λ₂ is the wavelength of the secondlight source. This explanation considers two light sources in order tosimplify the mathematics, in which the spectral intensity of the beamsprovided by each source is equal to I. λ₀ is the center wavelengthbetween the two wavelengths λ₁ and λ₂, and ω₀ is the center frequencybetween the frequency of the two sources. Δz is the axial distance ofthe object plane from the focal plane, and k is the wave vector of theillumination at the center frequency ω₀, where k=ω₀ /c=2π/λ₀. α is thehalf-angle of the objective lens aperture. The numerical aperture NA isNA=n sin α. The depth response, i.e., the square of demodulated signalfrom a planar reflector as a function of Δz, is as follows: ##EQU1## Thefirst term of this equation represents the confocal response, which ismodified by the second term, representing the field correlation functionprovided by the synthesized light source. The maximum improvement in thedepth response occurs if the sine function of the confocal response termand the cosine function in the field correlation term are oscillating atthe same frequency, i.e., if

    (λ.sub.2 -λ.sub.1)/λ.sub.0 =2 sin.sup.2 (α/2)(2)

then, the depth response becomes ##EQU2## which is twofold narrower thanthe confocal response at λ₀. The confocal response at λ₀ being Equation(1) absent the second term, cos[(λ₂ -λ₁)/λ₀ kΔz].

Since the field correlation function modifying the confocal response inEquation (1), cos(Δλ/λ₀ kΔz), is periodic, any mismatch between theoptical length of reference and image arm paths may be corrected withinthe confocal depth response, for example, within less than 20 μm. Theworst mismatch possible occurs if the length of the reference arm isΔz=3.2/(4k sin² (α/2)) away from the next optimum matching point wherethe interaction of the imaging and reference beams generate heterodynecomponents.

Referring to FIG. 3, the depth response using two light sources (N=2)with equidistant wavelength separation Δλ and five light sources (N=5)in synthesized light source 14 is shown as a function of the unit u,where u=4kΔz sin² (α/2). For an odd number N of light sources withequidistant wavelength separation in synthesized light source 14, thedepth response of Equation (3) is ##EQU3## The depth response narrows asN increases, i.e., as the number of different wavelengths which are usedincreases. The depth response of a single light source (N=1) is alsoillustrated in FIG. 3 for purposes of showing the narrowing depthresponse provided by using multiple, different wavelengths of light.

As discussed in connection with Table I, the improvement in depthresponse by using more than one light source is comparable to the effectof the increase of axial resolution by the use of higher NA confocaloptics in system 10. For example, if a single light source were used,instead of synthesized light source 14, at wavelength 820 nm withconfocal optics providing a NA of 0.2, the lateral resolution of system10 would be 3.32/(k sin α)≈2.2 μm and the axial resolution would be5.56/(4k sin² (α/2))≈18.0 μm. However, if the synthesized light sourceis used with two light sources at wavelengths 812 nm and 828 nm, theaxial resolution of the system can be reduced in half to 9 μm using thesame confocal optics. In this example, the reference and image arm pathlength mismatch may be corrected within a range of about ±10 μm with asensitivity of 1 μm.

The optimum NA for the confocal optics in system 10 using multiplewavelengths is determined by Equation (2) if the wavelength separationis of equal amount Δλ=λ₂ -λ₁. In other words, the wavelengths areequally separated from each other by Δλ. For synthesized light source14, the choice of NA should be such that the confocal response of theoptical system suppresses all except one of the peaks of the coherencefunction of the synthesized light source. The adjustment of the systemmay be easily facilitated by the wavelengths of the synthesized lightsource 14 being spaced equidistant from each other, thereby producing aperiodic coherence function. In this case, any peak of the coherencefunction can be chosen to coincide with the peak of the confocal depthresponse by adjusting the arm length mismatch between image arm andreference arm.

By utilizing synthesized light source 14 for illumination of object 12and detection at photodetector 37a of the heterodyne interaction of theimaging and reference beams, the performance of the system 10 in termsof axial resolution (and contrast) is improved beyond that limited bythe NA of the confocal optics of the system (which primarily is due tothe NA of objective lens 30). It is believed that the depthdiscrimination imposed by the temporal field correlation of thesynthesized light source 14 in combination with the axial resolution ofthe confocal optics improves the ability or resolution of the confocaloptics, enabling a user of the microscope system 10 to betterdistinguish cellular level tissue structures in the imaged section ofobject 12 on display 46.

From the foregoing description, it will be apparent that an improvedconfocal microscope system and method for confocal microscopy utilizingheterodyne detection has been provided. Variations and modifications ofthe herein described system and method and other applications for theinvention will undoubtedly suggest themselves to those skilled in theart. Accordingly, the foregoing description should be taken asillustrative and not in a limiting sense.

What is claimed is:
 1. A confocal microscope system for imaging sectionsof tissue below the surface of said tissue comprising:a light source forproducing a single beam of long coherence length light of differentwavelengths which represents the combination of a plurality of differentbeams of long coherence length light each of said different wavelengths;first optics for separating said single beam into an imaging beam and areference beam; an optical modulator for modulating the phase of saidreference beam; confocal optics for scanning and focusing said imagingbeam below the surface of said tissue and collecting returned light ofsaid imaging beam from said tissue; second optics for interacting saidreturned light with said modulated reference beam to provide a combinedreturn beam which has heterodyne components; a detector which receivessaid return beam and produces electrical signals corresponding to saidcomponents; and means for processing said electrical signals to producean image of said tissue section.
 2. The system according to claim 1wherein said single beam from said light source defines a coherencefunction having peaks in accordance with said different wavelengths, andsaid imaging beam transverses along a first path from said first opticsto said tissue and from said tissue to said second optics, and saidreference beam transverses along a second path from said first opticsthrough said optical modulator to said second optics, in which thedifference in length of said first path from said second path isapproximately equal to integer multiples of the separation of the peaksin said coherence function.
 3. The system according to claim 1 whereinsaid single beam from said light source defines a coherence functionhaving peaks in accordance with said different wavelengths, and saidcollected light of said imaging beam by said confocal optics defines aconfocal response having a peak, in which said second optics overlap oneof said peaks of said coherence function present in said reference beamwith said peak of said confocal response.
 4. The system according toclaim 1 wherein said confocal optics scan and focus said imaging beamalong a focal plane in said tissue, and said reference beam at saidsecond optics provides for spatially limiting the light of said returnbeam to a region of said tissue in said focal plane.
 5. The systemaccording to claim 1 wherein said confocal optics scan and focus saidimaging beam along a focal plane in said tissue, and said detectorprovides for spatially limiting the light of the return beam to a regionof said tissue in said focal plane.
 6. The system according to claim 1wherein said processing means further comprises means for demodulatingsaid electrical signals to obtain data representative of said image ofsaid tissue section.
 7. The system according to claim 1 wherein thedistance between the sources and the tissue, and the detector and thetissue, are approximately equal.
 8. The system according to claim 1wherein said different wavelengths are approximately equally spacedapart from each other.
 9. The system according to claim 1 wherein saidlight source comprises a plurality of light sources producing saidplurality of different beams at each of said different wavelengths, andthird optical elements for combining said plurality of beams into saidsingle beam.
 10. The system according to claim 9 wherein said image ofsaid tissue section has an axial resolution dependent on said differentwavelengths and the number of said plurality of light sources.
 11. Thesystem according to claim 1 wherein said confocal optics provide anumerical aperture below 0.7.
 12. The system according to claim 1wherein said scanning and collecting means comprises a deflector forscanning said imaging beam in at least two dimensions.
 13. The systemaccording to claim 1 wherein said first and second optics each representa beam splitter.
 14. The system according to claim 1 wherein said lightsources are laser sources.
 15. The system according to claim 14 whereinsaid laser sources are diode lasers.
 16. The system according to claim 1wherein said different wavelengths are each in the infrared spectrum oflight.
 17. The system according to claim 1 wherein said differentwavelengths number between two and five.
 18. The system according toclaim 1 wherein said imaging beam is scanned and focused by saidconfocal optics to an illumination spot of said different wavelengths.19. The system according to claim 1 wherein said detector comprises aconfocal aperture and said return beam is incident said confocalaperture before said detector.
 20. The system according to claim 1wherein said detector comprises a lens and said return beam is incidentsaid lens before said detector.
 21. A method for confocal microscopywhich images sections of tissue comprising the steps of:producing asingle beam of long coherence length light of different wavelengthswhich represents the combination of a plurality of different beams oflong coherence length light each of said different wavelengths;separating said single beam into an imaging beam and a reference beam;modulating the phase of said reference beam; scanning and focusing saidimaging beam below the surface of said tissue and collecting returnedlight from said tissue; interacting said returned light with saidmodulated reference beam to provide a combined return beam which hasheterodyne components; detecting said combined return beam to provideelectrical signals corresponding to said components; and processing saidelectrical signals to produce an image of said tissue section.
 22. Themethod according to claim 21 wherein said single beam defines acoherence function having peaks in accordance with said differentwavelengths, and said imaging beam transverses along a first path fromsaid first optics to said tissue and from said tissue to said secondoptics, and said reference beam transverses along a second path fromsaid first optics through said optical modulator to said second optics,in which the difference in length of said first path from said secondpath is approximately equal to integer multiples of the separation ofthe peaks of said coherence function.
 23. The method according to claim21 wherein said single beam from said light source defines a coherencefunction having peaks in accordance with said different wavelengths,said collected light of said imaging beam defines a confocal responsehaving a peak, and said interacting step further comprising overlappingone of said peaks of said coherence function present in said referencebeam with said peak of said confocal response.
 24. The method accordingto claim 21 wherein said scanning and focusing step scans and focus saidimaging beam along a focal plane in said tissue, and said method furthercomprises the step of spatially limiting the light of said return beamto a region of said tissue in said focal plane.
 25. The method accordingto claim 21 wherein said processing step further comprises the step ofdemodulating said electrical signals to obtain data representative ofsaid image of said tissue section.
 26. The method according to claim 21wherein said different wavelengths are approximately equally spacedapart from each other.
 27. The method according to claim 21 wherein saidstep of producing a single beam of light of different discretewavelengths is carried out with the aid of a plurality of light sourceswhich produce said plurality of different beams at each of saiddifferent wavelengths and optical elements for combining said pluralityof different beams into said single beam.
 28. The method according toclaim 27 wherein said image of said tissue section has an axialresolution dependent on said different wavelengths and said number ofsaid plurality of light sources.
 29. The method according to claim 21wherein said confocal optics provide a numerical aperture below 0.7. 30.The method according to claim 21 wherein said plurality of beams areproduced from laser sources.
 31. The method according to claim 30wherein said plurality of beams are produced from diode lasers.
 32. Themethod according to claim 21 wherein said different wavelengths are inthe infrared spectrum of light.
 33. The method according to claim 21wherein said different wavelengths number between two and five.
 34. Themethod according to claim 21 wherein said imaging beam is scanned andfocused to an illumination spot of said different wavelengths.
 35. Themethod according to claim 21 further comprising the step of passing saidcombined return beam through one of an aperture or lens before saiddetecting step is carried out.
 36. A system for imaging below thesurface of an object comprising:a confocal optical system for scanningsaid object and collecting light from said object; a plurality of lightsources operating at different wavelengths of long coherence lengthlight which are combined into a single beam for illuminating saidconfocal optical system; means for splitting said single beam into animaging beam for illuminating said confocal optical system and areference beam; means for modulating said reference beam; means forcombining said collected light from said object and said modulatedreference beam; means for detecting the portion of said combined lightrepresenting a section of said object to produce electrical signalsrepresentative of said section; and means for producing an image of saidsection of said object in accordance with said electrical signals.
 37. Amethod for producing an image of a section of an object using confocaloptics which scan and collect light from said object comprising thesteps of:combining into a single beam light from a plurality of beams ofdifferent wavelengths of long coherence length light; splitting saidsingle beam into an imaging beam for illuminating said object via saidconfocal optical system and a reference beam; modulating said referencebeam; combining light collected by said confocal optical system and saidmodulated reference beam; detecting the portion of said combined lightrepresenting said section of said object to produce electrical signalsrepresentative of said section; and producing an image of said sectionin accordance with said electrical signals.
 38. A system for imagingsections of tissue below the surface of said tissue comprising:a lightsource for producing a single beam of light of different wavelengths oflong coherence length light which represents the combination of aplurality of different beams of each of said different wavelengths;first optics for separating said single beam into an imaging beam and areference beam; a reference arm having an optical modulator formodulating the phase of said reference beam, in which said referencebeam travels along said reference arm along a reference arm path; animage arm having confocal optics for scanning and focusing said imagingbeam below the surface of said tissue and collecting returned light ofsaid imaging beam from said tissue, in which said imaging beam travelsalong said image arm along an image arm path; second optics forspatially overlapping said returned light with said modulated referencebeam to provide a combined return beam which has heterodyne components,wherein the difference in length of said image arm path and saidreference arm path between said first and second optics is in accordancewith said different wavelengths; a detector which receives said returnbeam and produces electrical signals corresponding to said components;and means for processing said electrical signals to produce an image ofsaid tissue section.
 39. The system according to claim 1 wherein saidconfocal optics have a numerical aperture and the tissue section imagedhas an axial resolution which is the same as that provided by otherconfocal optics having a higher one of said numerical aperture when usedwith a single wavelength beam.
 40. The system according to claim 1wherein said confocal optics define a numerical aperture, and saidtissue section imaged has an axial resolution in accordance with thenumerical aperture of the confocal optics, the different wavelengths ofsaid single beam, and the number of different beams providing saidsingle beam.
 41. The method according to claim 21 wherein said scanningand focusing step is carried out with confocal optics having at least anobjective lens through which said imaging beam is focused below thesurface of the tissue, said objective lens has a numerical aperture, andsaid tissue section imaged has an axial resolution in accordance withthe numerical aperture of the objective lens, the different wavelengthsof said single beam, and the number of different beams providing saidsingle beam.
 42. The system according to claim 1 wherein said detectorfurther comprises a photodetector and means for spatial limiting thereturn beam onto said photodetector.
 43. The system according to claim42 wherein said spatial limiting means is an aperture in which saidreturn beam passes through said aperture onto said photodetector. 44.The system according to claim 42 wherein said spatial limiting means isa lens through which said return beams passes to overfill saidphotodetector.
 45. The system according to claim 42 wherein said spatiallimiting means is provided by the interaction of said returned lightwith said modulated light.
 46. The system according to claim 42 whereinsaid spatial limiting means limits the returned light incident thephotodetector to a particular region of the tissue.
 47. The methodaccording to claim 21 further comprising the step of spatially limitingthe combined return beam before said detecting step is carried out. 48.A confocal microscope system for imaging sections of tissue below thesurface of said tissue comprising:a light source for producing a singlebeam of light of different wavelengths which represents the combinationof a plurality of different beams of each of said different wavelengths;first optics for separating said single beam into an imaging beam and areference beam; an optical modulator for modulating the phase of saidreference beam; confocal optics for scanning and focusing said imagingbeam below the surface of said tissue and collecting returned light ofsaid imaging beam from said tissue in which said confocal optics have anumerical aperture; second optics for interacting said returned lightwith said modulated reference beam to provide a combined return beamwhich has heterodyne components; a detector which receives said returnbeam and produces electrical signals corresponding to said components;and means for processing said electrical signals to produce an image ofsaid tissue section having an axial resolution in accordance with thenumerical aperture of the confocal optics, the different wavelengths ofsaid single beam, and the number of different beams providing saidsingle beam.
 49. A method for confocal microscopy which images sectionsof tissue comprising the steps of:producing a single beam of light ofdifferent wavelengths which represents the combination of a plurality ofdifferent beams of each of said different wavelengths; separating saidsingle beam into an imaging beam and a reference beam; modulating thephase of said reference beam; scanning and focusing said imaging beambelow the surface of said tissue and collecting returned light from saidtissue; interacting said returned light with said modulated referencebeam to provide a combined return beam which has heterodyne components;detecting said combined return beam to provide electrical signalscorresponding to said components; and processing said electrical signalsto produce an image of said tissue section having an axial resolution inaccordance with at least the different wavelengths of said single beamand the number of different beams providing said single beam.
 50. Asystem for imaging sections of tissue comprising:a light source forproducing a single beam of light of different wavelengths whichrepresents the combination of a plurality of different beams of each ofsaid different wavelengths; first optics for separating said single beaminto an imaging beam and a reference beam; an optical modulator formodulating the phase of said reference beam; confocal optics forscanning and focusing said imaging beam below the surface of said tissueand collecting returned light of said imaging beam from said tissue inwhich said confocal optics have a numerical aperture; second optics forinteracting said returned light with said modulated reference beam toprovide a combined return beam; a detector having an aperture whichreceives said combined return beam through said aperture to spatiallylimit the light of the combined returned beam to a focal plane in thetissue and produce electrical signals in accordance with said combinedreturned light; and means for processing said electrical signals toproduce an image of said tissue section.